The activities of many cellular proteins are determined by post-translational modifications. Phosphorylated forms of HEKZ/neu have been used to identify a subset of breast cancers with active signaling through the EGF pathway and aggressive clinical characteristics. Activity of the p53 tumor suppressor protein is regulated by a complex spectrum of post-translational modifications. Mass spectrometry using matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) provides a rapid and sensitive method to determine the functional status of p53 protein in tumors. To apply mass spectrometry procedures to archived tumor specimens, it will be necessary to develop methods to recover proteins from fixed, paraffin-embedded specimens. Specific Aim 1 will study the formation of crosslinks and adducts when purified p53 protein is exposed to two fixatives (formaldehyde or Histochoice). The extent to which the adducts and crosslinks can be removed will be tested by 4 methods. This provides a facile system to optimize conditions for cleaving crosslinks. Specific Aim 2 tests the effects of fixative on mammary epithelial cells expressing mutant or wild type p53 proteins. Methods for extraction and purification of p53 protein for analysis by MALDI-ToF will be optimized using this system. Specific Aim 3 utilizes mouse mammary tumors where the p53 status is known to validate the application of these methods to fixed, paraffin-embedded materials. Optimized procedures will be applied to human tumors to determine the patterns of post-translational modifications by MALDI-ToF.